The draft genome sequence of “Nitrospira lenta” strain BS10, a nitrite oxidizing bacterium isolated from activated sludge

The genus Nitrospira is considered to be the most widespread and abundant group of nitrite-oxidizing bacteria in many natural and man-made ecosystems. However, the ecophysiological versatility within this phylogenetic group remains highly understudied, mainly due to the lack of pure cultures and genomic data. To further expand our understanding of this biotechnologically important genus, we analyzed the high quality draft genome of “Nitrospira lenta” strain BS10, a sublineage II Nitrospira that was isolated from a municipal wastewater treatment plant in Hamburg, Germany. The genome of “N. lenta” has a size of 3,756,190 bp and contains 3968 genomic objects, of which 3907 are predicted protein-coding sequences. Thorough genome annotation allowed the reconstruction of the “N. lenta” core metabolism for energy conservation and carbon fixation. Comparative analyses indicated that most metabolic features are shared with N. moscoviensis and “N. defluvii”, despite their ecological niche differentiation and phylogenetic distance. In conclusion, the genome of “N. lenta” provides important insights into the genomic diversity of the genus Nitrospira and provides a foundation for future comparative genomic studies that will generate a better understanding of the nitrification process.


Introduction
Nitrification, the two-step oxidation of ammonia to nitrate via nitrite, is a key process of the biogeochemical nitrogen cycle. Nitrite-oxidizing bacteria (NOB) are chemolithoautotrophic microorganisms that catalyze the oxidation of nitrite to nitrate, the second step of the nitrification process. For decades NOB where considered as metabolically restricted microorganisms solely associated with nitrification. However, experimental findings contradict this opinion, indicating a versatile ecophysiology of many NOB [1][2][3][4] and highlighting their important role in and possibly outside of the nitrogen cycle [5].
The genus Nitrospira is the most diverse known NOB genus and is divided in six different phylogenetic sublineages [6][7][8]. Members of the genus are ubiquitously present in different natural and engineered ecosystems [5,[9][10][11]. Despite their high abundance, only eleven representative species, distributed within the six Nitrospira sublineages, have been obtained in enrichment or pure culture so far [7,8,[12][13][14][15]. Sublineage I and II Nitrospira are considered to be the dominant NOB in most wastewater treatment plants (WWTPs), playing a key role in the efficient removal of nitrogen via nitrification [6,16]. Besides their widespread distribution and crucial role, the physiology of Nitrospira species is highly understudied, mainly due to the lack of pure cultures and genomic data [3,17,18]. The recent identification of complete ammonia-oxidizing (comammox) Nitrospira [15,19] not only redefined the nitrification process, but also further indicated the importance of the genus and emphasized our poor understanding of the metabolic versatility present within this phylogenetic group.
"Nitrospira lenta" strain BS10 was isolated from a municipal WWTP [13] and it is the fourth isolate belonging to the sublineage II Nitrospira, besides N. moscoviensis [20], "N. japonica" [14], and the comammox organism "N. inopinata" [12]. Thus, insights into the "N. lenta" genome will shed light onto the genomic flexibility and metabolic diversity of the genus Nitrospira and aid further comparative studies between Nitrospira species.

Organism information
Classification and features "N. lenta" strain BS10 is a Gram negative, aerobic NOB isolated from activated sludge of a municipal WWTP in Hamburg, Germany (basic properties are summarized in Table 1) [13]. Based on 16S rRNA gene-based phylogentic analysis, "N. lenta" is affiliated with Nitrospira sublineage II but is only distantly related to the sublineage II type strain, N. moscoviensis (Fig. 1).
A pure culture of "N. lenta" was obtained after applying a combination of standard isolation methods (density gradient centrifugation and serial dilutions) with an optical tweezer system for the sorting of single cells [13]. "N. lenta" grows mainly planktonic and forms helical-shaped cells. Cells are 1.0-2.3 μm in length and 0.2-0.3 μm in diameter (Table 1, Fig. 2). As shown by Nowka et al. [13], "N. lenta" is able to grow at lower temperatures (10°C) than N. moscoviensis. Interestingly, while "N. lenta" is not able to tolerate high concentrations of nitrite, it exhibits a lower affinity for nitrite compared to N. moscoviensis and "N. defluvii", which indicates a clear niche differentiation among these Nitrospira species [21].

Genome sequencing information
Genome project history "N. lenta" was selected for whole genome sequencing on the basis of its relevance within the nitrogen cycle as well as due to the general lack of genomic information for Nitrospira species. Furthermore, because "N. lenta" was isolated from activated sludge, its genome was expected to yield insights that allow optimization and stabilization of the nitrification process in wastewater treatment. The draft genome sequence of "N. lenta" BS10 was completed on 27/07/2013. The high-quality draft genome is available in the European Nucleotide Archive (ENA) under study accession number PRJEB26290. An overview of the genome sequencing project is given in Table 2.
Growth conditions and genomic DNA extraction "N. lenta" was cultivated as described by Nowka et al. [13] in mineral salt medium amended with 0.02 g L − 1 NaNO 2 − as energy source. The cultures were incubated in the dark at 28°C, with moderate stirring (100-300 rpm). The genomic DNA was extracted following the hexadecyltrimethylammoniumbromide (CTAB) protocol provided by the DOE Joint Genome Institute (JGI, https://jgi.doe.gov/ user-program-info/pmo-overview/protocols-sample-prepa ration-information/) as described elsewhere [22].

Genome annotation
The draft genome of "N. lenta" was annotated using the MicroScope platform [24] as described in detail elsewhere [17]. The automatic annotation was manually checked and curated using the MicroScope Web interface MaGe [25]. The genomic features of "N. lenta" were compared to N. moscoviensis and "N. defluvii", the type strains of the Nitrospira sublineages II and I, respectively, using the OrthoVenn web service [26] for the identification and comparison of orthologous gene groups. Sequence similarities were calculated using an E-value of 1e-05. An inflation value of 1.5 was applied to generate the orthologous clusters.

Genome properties
The "N. lenta" draft genome consists of 22 contigs and has a total size of 3,756,190 bp with an overall G + C  The graphical circular map of "N. lenta" chromosome was generated using the CIRCOS software [33] content of 57.9% (Fig. 3, Table 3). From a total of 3968 predicted genes, 3907 (98.5%) and 61 (1.5%) are protein and RNA coding sequences, respectively. The genome encodes for 1 complete rRNA operon and 46 tRNAs, with 1 to 5 copies for each tRNA type. Moreover, 66.8% of the predicted genes were assigned into to Clusters of Orthologous Groups (COG) functional categories (Table 4).

Insights from the genome sequence
Nitrospira species belonging to sublineages I and II are the most abundant NOB in many environments and play a key role in N-cycling in engineered ecosystems [6,10]. Recent experimental data indicates a clear niche differentiation between sublineage I and II Nitrospira [21,27]. More specifically, "N. lenta", like other members of sublineage II, exhibits a lower maximum activity [21,27] and could be outcompeted by sublineage I Nitrospira at higher nitrite concentrations [28]. Despite their ecophysiological differences, sublineage I and II Nitrospira exhibit substantial genomic similarities. More specifically, "N. lenta" shares a core genome including 2223 orthologous protein clusters with N. moscoviensis and "N. defluvii". This corresponds to 67.3% of the pan-genome of the Nitrospira species included in this analysis (Fig. 4). Moreover, "N. lenta" features the lowest number of unique genes (1100, of which 51 are grouped in 18 paralogous protein clusters). Most of these unique genes lack any function prediction (Fig. 4).
In accordance with its ability to oxidize nitrite to nitrate [13], "N. lenta" encodes all proteins required for nitrite oxidation (Fig. 5), for which the key enzyme is a membrane-associated nitrite oxidoreductase (NXR) [29]. This protein complex belongs to the type II dimethyl sulfoxide reductase family of molybdopterin-binding enzymes and consists of three subunits [17,29]. The "N. lenta" genome contains two paralogous copies of nxrA and nxrB, encoding the NXR α and β subunits, respectively, and two copies of nxrC for the candidate γ subunit. Like all Nitrospira genomes analyzed to date, the "N. lenta" genome contains nirK, encoding the copper-dependent NO-forming nitrite reductase. While the function of NirK in Nitrospira is still unclear, a role in dissimilatory nitrite reduction is unlikely as no other genes involved in denitrification were identified in "N. lenta" or any other Nitrospira. One cannot exclude the possibility that NO plays a regulatory role in Nitrospira, for example in the regulation of forward versus reverse  The total is based on the total number of protein coding genes in the genome electron flow as proposed for Nitrobacter [30]. Moreover, "N. lenta" exhibits the genetic capacity for nitrogen assimilation from nitrite as its genome features nirA encoding the ferredoxin-dependent nitrite reductase. Interestingly, NirA is conserved in "N. defluvii", but not the other genome-sequenced sublineage II Nitrospira, which either encode an octaheme nitrite reductase [3,18], or, in the case of the comammox Nitrospira, lack assimilatory nitrite reduction pathways [15,19,31]. Interestingly, the "N. lenta" BS10 genome also features an ure operon encoding a functional urease (UreC), as well as a complete gene set (urtABCDE) for a high affinity urea ABC transporter. This implies that "N. lenta" is able to hydrolyse urea to ammonium and CO 2 , facilitating nitrogen and carbon assimilation from urea and reciprocal feeding between "N. lenta" and urease-negative ammonia-oxidizing bacteria [3,4]. The "N. lenta" urease is closely related to the enzyme of N. moscoviensis, but significantly differs from known ureases of ammonia-oxidizing bacteria [3]. "N. lenta" conserves energy by nitrite oxidation with oxygen as terminal electron acceptor. During nitrite oxidation catalyzed by NXR, two electrons are shuttled (putatively via cytochrome c) towards a putative novel bd-like terminal oxidase [17]. The proton motive force established through active proton pumping by this novel complex IV and/or the release of scalar protons during nitrite oxidation drives ATP synthesis by a F O F 1 -type ATPase (complex V). The other respiratory complexes (complexes I to III) will not contribute to energy conservation during lithoautotrophic growth on nitrite, but will operate in reverse to provide reducing equivalents for carbon fixation [17]. Moreover, the complete gene repertoire for the oxidative and reductive tricarboxylic acid (TCA) cycle is present in "N. lenta" for pyruvate oxidation via acetyl-CoA and CO 2 fixation, respectively. Moreover, the complete glycolysis/gluconeogenesis and pentose phosphate pathways were identified. The observed presence of glycolysis and the oxidative TCA cycle might indicate that "N. lenta" can benefit from a mixotrophic lifestyle in the presence of organic carbon, as has been reported for other Nitrospira representatives [6,16,32]. Finally, the "N. lenta" genome contains various defense mechanisms against heavy metals, antibiotics, and the antibacterial agent acriflavine. "N. lenta" encodes a superoxide dismutase for defense mechanisms against oxidative stress, as well as several bacterioferritins, which can participate in oxidative stress resistance mechanisms [17].

Conclusions
Together with N. moscoviensis and "N. japonica", "N. lenta" represents only the third cultured species of canonical nitrite-oxidizing Nitrospira from sublineage II. In this study, the genome of "N. lenta" was analyzed, demonstrating that "N. lenta" shares a significant amount of genomic features with other representatives of the genus. However,  [13,21] clearly suggest a niche differentiation between different species. The "N. lenta" genome will facilitate a better understanding of the metabolic versatility of the genus Nitrospira and will be useful for future comparative studies, especially those with a focus on species obtained from engineered systems.